This proposal aims to develop and exploit new molecules for measuring and/or manipulating neuronal messengers such as Ca2+, cAMP, cGMP, inositol-1 ,4,5-trisphosphate (IP3), and nitric oxide (NO). Various chemical and biological approaches will be pursued to improve the loading of fluorescent indicators for Ca2+ into adult brain tissue and to target indicators to specific sites within the cell. Infrared-fluorescing indicators for cytosolic Ca2+ will be improved for studies of signal transduction in photoreceptors, especially from Drosophila, and deeply buried neurons. New esters of IP3 that deliver IP3 across the plasma membrane will be improved and characterized. Such new techniques should help explore the possible roles of cytosolic Ca2+, stored Ca2+, cGMP, and IP3 in long-term depression (LTD) of parallel fiber synapses onto cerebellar Purkinje neurons. Because experiments with uncaged NO and CO have shown a precise coincidence requirement between Ca2+ and NO transients in inducing cerebellar LTD, the molecular mechanism of coincidence detection, the spatial spread of NO, and the importance of neuronal NO synthase will be investigated in cerebellar slices from normal and transgenic rodents. Another system of long-term plasticity to be explored is in limbic neurons such as from the locus ceruleus, where the relationship between opioid-induced acute and chronic changes in cAMP, channel modulation, and cAMP-regulated gene expression will be characterized by combined imaging of the cAMP indicator FICRhR, electrophysiological recording, and measurement of reporter gene expression.